Environmental microbial contamination results in losses of millions of dollars in equipment and product failure each year. Product deterioration, fouling, corrosion, adulteration, and diminished heat transfer capacity have all been attributed to microbial contamination. As an aid in monitoring and controlling detrimental microbial populations, there is a need for a simple sample processing technology which can be used in the field to concentrate and recover microorganisms.
Sulfate-reducing bacteria (SRB), by way of example, are among the most destructive environmental organisms. They cause corrosion and stress cracking of metals used in petroleum production and refining, (C. O. Obuekwe et al., Applied Microbiol. Biotechnol., 26, 389-393, (1987)) cooling water systems, (J. W. McCroy, in Microbiology of Cooling Tower Water, Chemical Publishing Company, New York, (1980)), waste treatment systems, (G. Kobrin et al., Waste Water Treatment Tank, Case Histories, compiled by R. E. Tatnall, Materials Performance, 20, 41-48, (1981)), pulp and paper production (A. Piluso, Materials Performance, 20, 41-48 (1981)). SRB produce hydrogen sulfide, which is more lethal than hydrogen cyanide. The sulfide products can also be used by other organisms to manufacture sulfuric acid, (P. Bos et al., Microbiology of Sulfur-Oxidizing Bacteria, in The Metal Society, 18-27, (1983)), which in turn can rapidly destroy concrete municipal sewage systems. Infestation of waterways by SRB organisms, (J. R. Postgate, in The Sulfate-Reducing Bacteria, 2nd Edition, Cambridge University Press, New York, (1984)), is becoming a serious problem, being both a symptom of oxygen depletion and a problem because of generation of toxic hydrogen sulfide. The combined effect of oxygen depletion and toxic concentrations of hydrogen sulfide suppresses marine life.
Until recently, microbiological detection of environmental microorganisms was limited to laboratory culture. Generally, culture methods are slow, requiring days or weeks to accomplish and are not suited to field testing where control of the organisms is needed. Culture methods have thus been of limited value in monitoring and controlling microbial populations. Many newer direct detection methods developed to accelerate or eliminate culture methods are also fraught with difficulties because of sample complexity, microorganism diversity and analytical insensitivity.
Environmental samples such as soil, sewage, surface water and, particularly, crude oil are complex mixtures which are heterogeneous in both their solid and soluble components. In composition, the samples sometimes contain high concentrations of solid sediments, colloids, emulsions, soluble and insoluble salts, biopolymers, biodegraded debris, inert materials and contaminating industrial and natural chemicals. The heterogeneity and complexity of environmental samples can prevent or invalidate direct detection methods. Microorganisms can adhere to particulates, rendering the microorganisms inaccessible to detection by conventional approaches which rely on liquid filtrate samples. Also chemicals and solutes contained within the samples can interfere or inhibit test methods. Sample coloration, for example, can obstruct test results when colors are part of the assay.
Direct detection of microorganisms and/or their products is further complicated by the fact that in many instances, sample materials contain only small numbers of microorganisms (10.sup.2 to 10.sup.5 cells/gm). Furthermore, in many cases the microorganisms do not comprise a single strain or even a single genus but are a diverse collection of many different types of organisms. For these reasons, direct detection requires highly sensitive methods possessing broad spectrum specificity or a means of selecting specific organisms as described in U.S. Pat. No. 4,592,994. Because of the requirements of both high sensitivity and broad spectrum specificity, direct detection approaches such as immunoassays directed toward the cells or their products are subject to chemical interferences.
To circumvent these problems, some manner of sample treatment has been typically required to concentrate the small numbers of organisms present in field sample materials and to free them from interfering materials which prevent analysis or cause inaccurate or false results. Generally, treatment processes can involve centrifugation, membrane filtration or chemical precipitation steps.
Chemical treatment, diatomaceous earth (DE) filtration, and charcoal adsorption are used extensively for drinking water treatment and waste water processing (Linstedt et al., Water Research, 8 753-760 (1974). Studies of these large scale production processes have shown that the processes are effective in screening debris, grit removal, clarification and biofiltration. Depending on the plant operating parameters and efficiency, the resulting water filtrates can be essentially free of particulates and microorganisms.
Pilot plant tests reported by K. P. Lang'e et al., (J. Am. Water Works Assoc., 78, 76-84 (1986)), have shown that virtually 100% of the parasite Giardia lamblia cysts are removed by filtration through DE over a wide range of conditions. Smaller organisms, such as coliform bacteria can also be removed but removal is functional and dependent on the grade of DE. Diatomaceous earth filters have also been reported to remove heterotrophic bacteria from sea water (J. Illingworth, et al., Aquaculture, 17, 181-187 (1979). With the aid of chemical agents and coated DE, virus can also be filtered. R. De Leon et al., Wat. Res., 20, 583-587 (1986), showed that rotoviruses and f2 coliphages could not be removed by strictly mixed media filtration. However, the addition of small amounts of alum and coagulant polymer improved the effectiveness of viral removal. T. S. Brown et al., J. Am. Water Works Assoc. 66, 98-102 (1974), also confirmed the removal of viruses from water by DE filtration by removing T2 bacteriophages and polioviruses from it. However, the conditions for removing these viruses appeared to be virus specific and depended on the appropriate adjustment of pH and the selection of an effective DE coating.
Generally, DE filtration is applicable for waters that do not contain high concentrations of sediment or algae, both of which are likely to cause "binding" of the filter media resulting in increased back pressure and obstructed fluid flow. DE biofiltration by itself has thus not been applicable for samples containing high sediments content or mixed phase samples such as water/oil emulsions. (K. P. Lang'e et al., J. AWWA, 78, 76-84 (1986). For these reasons, DE biofiltration technology has been limited in practice to large industrial and municipal plants where consistency over water composition can be anticipated and careful control over the hydrodynamics of fluid flow and the DE bed parameters can be maintained. Such filtration is substantially useless in any attempt to isolate SRB from oil well samples; plugging of the filter occurs too quickly.
The primary mechanism of DE biofiltration is straining. Therefore, biofiltration processes must be carried out slowly with care being taken not to disturb the pore structure of the primary DE bed. This requires extensive equipment to provide control over the fluid hydraulics of the filtration process. Such equipment is impractical for analytical testing and field use.
A secondary mechanism of DE biofiltration is the adsorption of fine particles or colloids on which the microorganisms are secondarily adsorbed. The surface charges on the DE and the colloids in the water samples limit the efficiency of this process.
The use of chemicals added either directly to the water or in combination with DE filtration also has been employed to circumvent the difficulties of biofiltration processes. This physical/chemical treatment process relies on chemical coagulation followed by sedimentation to remove the suspended solids. Typically the use of aluminum or iron salts as coagulants achieves significant phosphorous and particle removal. Linstedt et al., Water Research, 8, 753-760 (1974), showed that the addition of alum and other chemical agents can effectively remove soluble phosphates, turbidity, bacteria and heavy metals from wastewater. Lee et al., Lab. Pract., 23, 297-298 (1974), demonstrated that chemical flocculation with aluminum potassium sulfate can be used efficiently to recover bacteria from cell culture fluids by sedimentation.
While chemical flocculation is an easily accommodated procedure, it is generally not used to harvest microorganisms for analytical determination. Flocculation processes are quantitatively dependent on concentration of flocculating agent, the amount of competing charged species present in the sample and the charge on the microorganism. If sample materials vary widely in the concentration of competing materials, the microorganisms can not be recovered reproducibly. Also, too much flocculating agent can prevent flocculation. Furthermore, flocculation reagents can react with or prevent the reaction of reagents required for the further analysis of the sample. Most importantly the flocculating agents can solubilize or alter the chemical composition of cells. In this way, the coagulation reagents can compromise the immunological reactivity of the microorganisms. For these reasons, the use of chemical additives has not been an effective means for collecting and recovering microorganisms and removing contaminating solutes from them.
U.S. Pat. No. 4,515,697 describes a method for flocculating microalgae or other organic particles without the addition of reagents. The method passes the solutions containing the microalgae or other organic particles which they wish to collect through a fixed granular layer at a rate which allows the microalgae or other organic particles to collect and flocculate, clogging the bed of granular material in the process. They disclose the use of sand, ceramics and particles of calcinated clay as the granular material. There is no indication how quantitative the collection is.
Likewise, WO 87/00199 discloses that diatomaceous earth has the ability to irreversibly entrap microbes or the like within the interior structure of the particle and uses diatomaceous earth to concentrate bacteria on fibrous materials. This enabled the bacteria to be cultured and enhanced biological and chemical reactions by them due to their concentration. The efficiency of the recovery of the bacteria was not disclosed.
And GB 2,189,317 describes the use of titanous hydroxide suspension to immobilize sulfate-reducing bacteria so that they could be detected by an ELISA method.
It is an object of the present invention to provide an inexpensive method to collect and recover microorganisms from environmental samples. The method must be capable of use in the field to facilitate the direct detection of microorganisms and (or) their products. It is a further object to provide the means for concentrating the organisms and freeing them of interfering solutes for immunoassay or other detection. It is a still further object to provide a means including apparatus to make use of inexpensive and commonly available biofiltration materials to accomplish the foregoing objectives.